We propose to study the mechanisms involved in the replication of hepatitis B-type viruses and the relationship between human hepatitis B virus (HBV)-infection and hepatocellular carcinoma, using the techniques of molecular biology and a newly discovered animal model for HBV, the ground squirrel hepatitis virus (GSHV). Specimens obtained by open liver biopsies and autopsies from ground squirrels infected in captivity will be characterized by molecular hybridization using cloned GSHV DNA and by immunological procedures for virus-specific macromolecules. We will grow cells from infected livers in short term cultures in order to analyze synthetic pathways for viral DNA, RNA, proteins, and particles; we will also attempt to infect cultured cells from uninfected animals to study early events in viral replication. To define the biochemical determinants of infectivity, we will chemically alter virus particles prior to infection of squirrels, and we will test the possibility that cloned, closed circular GSHV DNA is infectious when appropriately delivered to animals. If so, we will construct mutant and recombinant genomes which will be assayed in animals to define the genetic determinants of infectivity and pathogenicity. These studies will be complemented by detailed mapping of the GSHV genome and by the examination of virus strains and squirrels obtained at several locations in California. To examine gene regulation at a finer level, we will use a fully sequenced, cloned genome of HBV to make mutant and recombinant molecules; the expression of these will be examined in mouse L cells after DNA transformation and by RNA synthesis in a cell-free system. Lastly, a series of tumor samples from patients in Hong Kong with hepatocellular carcinoma will be tested for the presence of integrated and unintegrated viral DNA and for its expression as RNA and protein. We will also test these samples for two phenomena of current interest in retrovirus-induced cancers: the capacity of tumor DNA to initiate morphological transformation of cultured mouse cells and the activation of cellular genes situated near integration sites occupied in multiple tumors.